protein corona formation for nanomaterials and proteins of a similar size: hard or soft corona?

Nanoscale

PAPER

aCEREGE (UMR 7330 CNRS/Aix Marseille

L'Arbois, BP 80, 13545 Aix en Provence, FrabGDRi iCEINT International Consortium

Nanotechnology, www.i-ceint.orgcCEA/DSV/iBEB/SBTN, Laboratoire d'Etude

Bagnols sur Ceze Cedex, France. E-mail: cla

† Electronic supplementary informationsterile CeO2 NP suspension preparation bdata from fractional analysis of chroanalysis of the structure of CeO2 in contameasurement of NPs in different10.1039/c2nr33611a

Cite this: Nanoscale, 2013, 5, 1658

Received 17th July 2012Accepted 4th December 2012

DOI: 10.1039/c2nr33611a

www.rsc.org/nanoscale

1658 | Nanoscale, 2013, 5, 1658–166

Protein corona formation for nanomaterials andproteins of a similar size: hard or soft corona?†

Wei Liu,ab Jerome Rose,ab Sophie Plantevin,c Melanie Auffan,ab Jean-Yves Botteroab

and Claude Vidaud*bc

Nanoparticles (NPs) entering a biological fluid undergo surface modification due to dynamic,

physicochemical interactions with biological components, especially proteins. In this work we used

complementary bio-physico-chemical approaches to characterize the effects of interactions between

CeO2 NPs, immunoglobulins (IgGs) and bovine serum albumin (BSA) of a similar size on protein

structural evolution as well as formation of (hetero-) aggregates. Using circular dichroism we showed

that IgGs and BSA underwent significant structural changes after interaction with NPs. The NPs and

protein–NPs were observed after size exclusion chromatography, highlighting the fact that few

aggregates were stable enough to pass this mild separation step. X-ray absorption spectroscopy

suggested that the surface chemistry of NPs was not affected by these proteins, also implying weak

interactions. Competitive experiments revealed that, while the serum was more concentrated for BSA,

IgG–NP aggregates were more stable. Altogether, our results indicate that, under our experimental

conditions, the formation of a “protein corona” is an unstable and reversible mechanism. This indicates

that, when NPs and proteins are similar in size, the adsorption concept (i.e. protein corona concept)

cannot be applied to model the NP–protein interaction, and a heteroaggregation model is more

appropriate.

Introduction

CeO2 NPs show growing and prospective uses in medicalapplications, cosmetic products, polishing materials andbyproducts from automotive fuel additives, according to theirspecic, catalytic, light properties.1 However, their toxicitiestowards different eukaryotic or prokaryotic cell lines have beendemonstrated by several studies.1–6 Other studies using bareand coated CeO2 NPs have highlighted the role of size andsurface properties in their cellular uptake.7,8

Indeed, when nanoparticles enter a biological uid, theyundergo surface modication due to dynamic physicochemicalinteractions with biological components, especially proteinadsorption.9–13 The composition and structure of the protein-adsorbed layer on NPs, namely the “protein corona”, depends

universite), Europole Mediterraneen de

nce

for the Environmental Implications of

des Proteines Cibles, BP 17171, 30 207

[email protected]

(ESI) available: Details of control ofy DLS; the uorescence measurementmatographic elution; EXAFS spectract with BSA and IgG; the zeta potential

experimental media. See DOI:

8

on the composition of the medium.4 This has been exempliedby studies using Roswell Park Memorial Institute (RPMI) andDulbecco modied Eagle's (DMEM) media.14 The protein–NPcomplexes interact immediately with living systems and affectthe biological response in in vitro experiments. It is well estab-lished that interactions with serum modify NP behavior andeffects. For example, the internalization of polysaccharide (PS)–NPs into bronchial epithelial cell lines is reduced in the pres-ence of serum,15 and it has also been noted that serum protectsmacrophages against PS–NP toxicity.16 Therefore, characteriza-tion of the interaction between NPs and identied proteins iskey to understanding their biological effects.

Professor Dawson's group of collaborators introduced theconcept of a “hard protein corona” ve years ago.17 In their mostrecent papers, Milani et al.18 proved that the protein corona canbe composed of different protein layers, with the rst beingirreversibly xed while the outer layers are less strongly xedand can be exchanged (“so” corona). These authors success-fully modeled NP–protein interactions using adsorptionconcepts (NPs being the adsorbent, proteins being the adsor-bate) and, more specically, Langmuir adsorption formalism.All these crucial discoveries can be compared with the generalliterature concerning the adsorption of natural organic matteronto particles, to which the adsorption theory can alsobe applied and in which ‘hard’ (¼ irreversible) and ‘so’ (¼reversible) layers are also formed. The adsorption model

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implies that the size of the adsorbed molecule (adsorbate) ismuch smaller than that of the substrate (adsorbent). In themajority of recent papers, the nanomaterial size was not lessthan 40 nm, thus agreeing with this hypothesis (except inLundqvist et al.19 (6 and 9 nm SiO2)). However, when theadsorbate and the adsorbent are of similar sizes, i.e., for NPssmaller than 10 nm, the concepts behind the adsorptionphenomenon reach their limit of validity. The surface energy atthe origin of the adsorption of molecules is balanced by shearforces due to Brownian motion, and can lead to detachment.One strong objective of our work was, therefore, to determinewhether adsorption concepts can be applied to systems exhib-iting NPs and proteins of a similar size.

The main protein source in a cell culture medium isprovided by fetal calf serum (FCS). One of the characteristics ofFCS is its variability in protein composition. Nevertheless,albumin and immunoglobulins (IgG) are by far the mostabundant proteins in FCS-containing cell culture media and areoen suspected to be potential targets of NPs. Many studieshave demonstrated the binding of these proteins to bare orcoated NPs, and modication of the protein conformation inthe presence of NPs. However, although studies have qualiedNP–protein interactions, details regarding the quantication ofNPs and proteins in the complexes formed, as well as the effectsof complexation on the surface chemistry of NPs and/or proteinstructure, remain poorly dened.

In our study, the interaction of CeO2 NPs with isolatedbovine serum albumin (BSA) and IgG in buffer solutions wasrst shown by circular dichroism (CD) studies, which alsoindicated structural modications for both proteins. X-rayabsorption spectroscopy suggested that the surface chemistry ofnano-CeO2 is not affected by interaction with BSA and IgG. NPdistribution within the protein population of the DMEM-F12-FCS medium was analyzed by size exclusion chromatography.This mild separation method minimizes the disturbance ofcomplexes at equilibrium. Fluorescent-labeled BSA and IgGwere used to spike the chromatographic step and evaluate theirrelative affinities for the NPs. The analysis of NP distribution ineach protein fraction was performed by determining theirincidence as protein uorescence and comparing this to theirNP content evaluated by ICP-MS. The relative affinities of BSAand IgG were then evaluated through competition by uores-cence spectroscopy. The results highlighted low affinity and theabsence of NP corona. The results are discussed with regard tothe possible different impact of NPs on cell toxicity.

ResultsPart 1: CeO2 NP–protein interactions

Effects of biological buffered solutions on nano-CeO2 homo-aggregation. To avoid any precipitation and aggregation of theproteins, and ensure similar stability and monodispersity of theNPs, the 8 mM Tris–HCl pH 7.4 buffering solution was selectedas a compromise for observing NP–protein interactions. Basedon DLS, we observed that BSA (6 mM) and IgG (2 mM) remainedmonodispersed in Tris–HCl with an average size value of 9 nmand 7 nm for IgG and BSA respectively (Fig. 1A and C). BSA and


IgG concentrations and the concentration ratio were selectedwith respect to BSA and IgG concentrations in the nutritivemedium tested in the following parts.

While nano-CeO2 is mono dispersed in the stock solution(Fig. SI-1†) with an average hydrodynamic radius of 7 nm and anarrow distribution, CeO2 NPs strongly aggregate in the protein-free Tris–HCl buffer. At 10 mg ml�1 of NPs, two size fractions ofaggregates are formed with average hydrodynamic sizes of 100and 500 nm respectively. At 60 mg ml�1, much larger homo-aggregates of NPs are formed with the smallest size fraction at300 nm and the largest even greater than the DLS detectionlimit (several microns). When CeO2 NPs are mixed with proteinsthe aggregates are smaller which indicates that CeO2 nano-particles interact with the proteins to form hetero-aggregates. Inthe case of IgG at 10 mg ml�1, the particle size remains verysmall with an average size of 10–12 nm indicating a very gooddispersion of CeO2 NPs (Fig. 1B). Such a small size suggests thataggregates are mainly composed of one CeO2 NP and one or veryfew proteins. At higher concentrations two main narrow peaksare observed: the sizes of aggregates increase with two sizefractions ranging between 45–50 nm and 550–600 nm at 20 and40 mg ml�1, and reached 450 nm at 60 mg ml�1 (Fig. 1B). In thecase of BSA (Fig. 1C) the size distributions of aggregates arebroader compared with IgG. At 10 mg ml�1 there are two sizefractions with an average size of 45 and 350 nm respectively.When the CeO2 NP concentration increases (20 and 40 mg ml�1)again there are two size fractions in the 30–700 nm range whileat the highest concentration tested (60 mg ml�1) there is onefraction with a peak value at 250 nm. The difference between thesize distribution of IgG and BSA in the presence of CeO2 NPsmay be due to different interactive forces.

Impact of CeO2 NPs on protein conformations. CD analysesprovide additional information regarding modication of thestructural conformation of the proteins20 interacting with theNPs. CD spectra measurements were carried out at roomtemperature using constant concentrations of BSA or IgG withincreasing concentrations of NPs (Fig. 2). Control experimentsconrmed that the 10 and 60 mg ml�1 NP suspensions did notpresent any dichroic spectra.

The BSA contains predominantly a-helices as secondarystructural elements, giving two negative bands at 208 and220 nm in the CD spectra (Fig. 2A). In the presence of NPs, theintensity of these bands decreased and an isodichroic point wasobserved at 203 nm (Fig. 2A). The isodichroic point indicatesthe presence of two-state dichroic models (bound and unboundstates) during the interaction with NPs. These results highlightthe fact that BSA–NP interactions exist, and modify the BSAsecondary structural elements (a-helices).

The IgG secondary molecular structure predominantlycomprises b-sheets, with a minimum signal intensity at 218 nm.Again, the IgG CD spectra were modied as the amount of NPsincreased: the intensity of the 218 nm signal decreased and anisodichroic point was observed at 208 nm (Fig. 2B). As for BSA,this result conrmed the fact that IgG interaction with NPsoccurred and altered its secondary structure. However, the struc-tural changes in IgG were more pronounced than for BSA: for anequivalent amount of NP versusmolar concentrations of proteins,

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Fig. 1 Colloidal stability of IgG, BSA and NPs respectively (A) and in the case of IgG (B) or BSA (C) mixed with NPs (0, 10, 20,40, 60 mg ml�1). Working buffer: 8 mMTris–HCl pH 7.4.

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a 30% decrease in signal was obtained at 40 mg ml�1 NPs for IgG,whereas only a signal decrease of only 15% occurred for BSA.

By assuming that the secondary structure of proteins canonly be modied when they were in contact with CeO2 NPsurfaces, a simple calculation indicates that the number ofproteins associated with one NP lies in the 9–11 range for IgG aswell for BSA.

To determine whether NP–protein interactions affect thesurface structure (crystalline stability) and chemistry (redoxstate stability and Ce surface site complexation) of CeO2 NPs,X-ray absorption spectroscopy (XAS) at the Ce LIII edge was

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performed. Indeed it was shown previously that CeO2 NPs caninteract with nutritive media; the resulting effect was thesurface reduction of Ce4+ to Ce3+ mainly due to interaction withthiol groups of thiolated molecules such as protein.2 Thecomparison between the XAS spectra of CeO2 NPs before andaer interaction with IgG or BSA revealed no major structuralchanges nor reduction of Ce4+ atoms at the surface of the NPs.Based on XAS and CD analysis we estimate that the majority ofNP–protein interactions occurred through physical interaction(electrostatic interaction, steric stabilization.) rather thanchemical surface complexation.


Fig. 2 Evolution of circular dichroism spectra of BSA (A) and IgG (B) after contact with different concentrations of CeO2 NPs. Experimental conditions: 8 mM Tris–HCl,pH: 7.4 buffer, BSA (3 mM) or IgG (2 mM) were mixed with 10, 20, 40, 60 mg ml�1 of CeO2 NPs.

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Competition between BSA and IgG for interaction with CeO2

NPs. Competition studies were performed between BSA and IgGfor NPs binding in the buffer. Alexa� 488-labeled BSA (3 mM)and Alexa� 647-labeled IgG (1 mM) were prepared in 8 mM,Tris–HCl pH 7.4 buffer. The excitation wavelengths of labelledBSA and IgG were xed at 488 and 647 nm and emissions weremonitored at 521 and 674 nm, respectively (Fig. 3). The inter-action of NPs with each protein could be distinguished bycomparing uorescence intensities due to the different uo-rescent dyes conjugated to each protein. We checked that thecovalent labelling and associated biochemical modicationsdid not modify protein–NP interactions by measuring the effectof NPs on tryptophan uorescence at 347 nm on the labeled andunlabeled proteins (data not shown).

Evolution of the uorescence spectra for each protein ispresented in Fig. 3. Following the gradual addition of NPs, theuorescence emissions of the labelled BSA or IgG were stronglyquenched, conrming the interactions between the NPs andBSA or IgG.

Fluorescence intensities (I) were determined at the lmax

emissions and the results were compared with the initial


uorescence of individual proteins (I0). Data were analyzedusing the Stern–Volmer representation (Fig. 4). In all cases, I0/Ivaried linearly over NP addition, and could be expressed as 1 +Ksv [NPs], where Ksv is the Stern–Volmer constant. In the case ofstatic uorescence quenching, Ksv equals the associationconstant Ka. The slope obtained for BSA (Fig. 4A) is 1.66-foldhigher for BSA than for IgG (Fig. 4B), indicating a higher asso-ciation constant. When NPs were exposed to both proteinssimultaneously, the presence of IgG strongly altered BSA uo-rescence, as observed by the decrease in slope. However this wasnot observed in the reverse situation. This led to the conclusionthat BSA–NP complexes can be dissociated in the presence ofIgG, while IgG–NP complexes persist in the presence of BSA.These results highlight the more stable interactions betweenNPs and IgG than between NPs and BSA.

Part 2: behaviour of CeO2 NPs in a biological medium

Colloidal stability of CeO2 NPs in a cell culture medium.DMEM-F12 complemented with FCS (fetal calf serum) isa commonly used medium for cell culture experiments. The

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Fig. 3 Fluorescence emission spectra of Alexa� 488-labeled BSA (3 mM, solid line) and Alexa� 647-labeled IgG (1 mM, dashed line) with increasing concentrations ofNPs: (1) 0, (2) 10, (3) 20, (4) 30, (5) 40, (6) 50 and (7) 60 mg ml�1.

Fig. 4 Competition studies between labelled BSA and IgG for NP binding. TheStern–Volmer representation of fluorescence variations vs. increasing NP concen-trations. (A): Effect of IgG (1 mM) on BSA (3 mM) fluorescence (dashed line) andcompared with BSA fluorescence alone (solid line). (B): Effect of BSA (3 mM) on IgG(1 mM) fluorescence (dashed line) and compared with IgG alone (solid line).

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NPs–protein interactions were then assessed in this complexmedium. We rst checked the absence of large molecules in thepure DMEM-F12 medium (no FCS nor NPs). No DLS signal wasobtained, conrming that no large molecule existed (data notshown). When FCS was added to the DMEM-F12 (without NPs),a size distribution ranging from 5 to 15 nm with a peak value at6.5 nm appeared, in agreement with the size of the FCS proteins(Fig. 5).

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When CeO2 NPs were added to the FCS-free DMEM-F12 (i.e.w/o proteins), the size of aggregates was greater than 5–10microns and therefore outside the DLS size measurementrange. This strong aggregation (homo-aggregation) is related tothe pH being close to the CeO2 NP IEP (iso-electric point) andthe high ionic strength (larger than the critical concentration ofcoagulation).

When CeO2 NPs were added to the DMEM-F12 com-plemented with FCS, the aggregate sizes decreased drastically(Fig. 5). In such a complex medium, the mean size remainsbelow 20 nm for 10 mg l�1 of NPs, and two size classes wereobserved (mean size 15–38 nm and 600 nm) for 60 mg l�1 of NPs.This important fall in hydrodynamic diameter is a direct proofof NP–protein interactions. This corroborates the trends alreadyobserved in the Tris–HCl buffer (part 1, Fig. 1).

However, the aggregation kinetics in the DMEM-F12-FCS wasrather slow. The size distribution over time for NPs at 10 mgml�1 (Fig. 5A) showed a slow and asymmetrical increase. Aer 2hours in the presence of NPs, the size distribution of the entitiesformed in the DMEM-F12-FCS remained unchanged whichindicated that interaction between NPs and proteins occurredslowly. Aer 6 hours, the mean size of aggregates increasedfrom 5–6 nm to 16 nm. This corresponds to the hetero-aggregation process. Between 6 and 24 h, a slight redispersionwas observed (mean size at 13 nm), possibly due to the temporalevolution of the components constantly rearranging within theaggregates.

The addition of 60 mg ml�1 of NPs to the DMEM F12-FCSsolution led to an increase of scattered light intensity due toparticle fractions with a mean hydrodynamic radius of 7 nm.This increase could correspond to dispersed proteins from theFCS and added CeO2 NPs that remained monodispersed.However a fraction of NPs interacted with proteins because aclass of particles was detected around 25 nm. Aer 2 h, 6 h and24 h, the heteroaggregation led to aggregates with hydrody-namic diameters centered on 15, 25 and 38 nm respectively(Fig. 5B). Moreover, aer 6 h of contact, a second class of


Fig. 5 Colloidal stability of NP suspensions in the DMEM-F12-FCS culture medium at 10 (A) and 60 mg ml�1 (B) after 1 min, 2, 6 and 24 h incubation time.

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particles appeared, with an average size of 450 nm, reaching550 nm aer 24 hours.

CeO2 NP distribution in the culture medium. We previouslydemonstrated that NPs interact with isolated BSA or IgG inbuffer solutions and also with proteins in DMEM-F12-FCS asrevealed by the size distribution analysis. We also highlightedthe fact that the interactions between NPs and IgG were prob-ably more stable than the ones between NPs and BSA. Sizeexclusion chromatography was performed to evaluate the effectof NPs on the protein populations in the culture medium andalso to assess the reversibility/irreversibility of the NP–proteininteractions.

To help locate IgG and BSA in the eluted fractions, uores-cent IgG and BSA were spiked in the DMEM-F12-FCS culturemedium before the chromatographic processes. The twolabeled proteins were added to the medium without intro-ducing any dilution effect and in proportions mimicking theirconcentrations in the serum. The protein labeling was opti-mized to reach the same range of absorbance and uorescenceso as to avoid any different sensitivities in our measurements.The labeled proteins were also used as probes to measureuorescence variations in corresponding fractions of thecontrol run (without NPs) and the test run (with NPs).

Several control experiments were performed prior to sizeexclusion chromatography to check for possible artefacts.Because the NPs might be adsorbed at vial surfaces during thecontact between NPs and the medium, we quantied theamount remaining in suspension (ICP-MS analysis). The NPconcentration decreased from 60 to 41 mg ml�1, indicating 22%of nano-CeO2 sorption on the surfaces, as no settling occurred.

In the test run, 60 mg ml�1 NPs were incubated in DMEM-F12-FCS, for 20 h at room temperature, before injection into thecolumn, whereas an equivalent volume of buffer was added forthe control run. The chromatographic proles were obtained bymonitoring the absorbance variations on-line at 280 nm (allproteins), 488 nm (labeled BSA) and 647 nm (labeled IgG)(Fig. 6). The eluted fractions were collected (4 ml) for further off-line analysis: full UV-vis absorbance spectra, uorescencespectra and ICP-MS analysis. Both chromatograms of the testand control runs are presented in Fig. 6.

The absorption variations registered at 280 nm displayedthree main peaks. The rst main A peak corresponds toproteins. The absorption at 647 nm and 488 nm indicated thefractions for which the labelled IgG (150 000 Da) and BSA


(66 000 Da) were eluted, indicating an overlap in the elutions ofthese proteins. Therefore, the strong shoulder observed in thelehand part of the A peak (A0 to A7 fractions) corresponded tothe elution of very large proteins or protein aggregates (probablymore than 300 000 Da). It is worth noting that large proteins orprotein aggregates only represent minor amounts of the overallprotein populations. The other two fractions (B and C) exhibitedabsorbance only at 280 nm, related to low molecular weightproteins/molecules.

When CeO2 NPs were added to the DMEM-F12-FCS mediumand injected into the column, the chromatogram showed analmost identical prole to that of the control run. A slight fall insignal was observed, whatever the monitored wavelengths, butit was within the experimental variations.

The absorption and uorescence spectra were thereforescanned for all the collected fractions (Fig. 5, ESI†). Fluores-cence analysis was performed on the fractions for which thelabelled proteins were eluted (ESI, Table S1†). The results of thenormalized uorescence variations between the two runs (I0:uorescence of the fractions in the control run, I uorescence ofthe corresponding fraction in the test run) are reported in Fig. 7.As stated above, IgGs were predominant in fractions A8–A10. Infraction A11, both proteins were eluted, then the IgG populationwas less concentrated in fractions A12–A14, while BSA waspredominant in fractions A13–A14.

We rst observed that, except for the A13 fractions, BSAuorescence variations were less important than those of IgG.Conversely, IgG uorescence was modied when NPs werepresent in the medium. These results may have indicated amore important NP interaction with IgG than with BSA, thusconrming the results obtained in buffered solutions (part 2).

However, the NP recovery estimated from the ICP-MS results(Fig. 8) was low (z6%). We hypothesized that CeO2 NPs mightbe adsorbed onto the gel phase as was previously mentioned forthe vial. This strong adsorption onto the gel was conrmed bymimicking the run without adding FCS to the DMEM-F12. Inthis case, the NPs did not elute at all, even aer washing withseveral column volumes. Firstly, this indicates that FCSbiomolecules are necessary for NP elution. Secondly, the gelsurface and the multiple exchanges between the proteins andthe gel probably led to destabilization of the NP–proteinheteroaggregates.

Thus, only strong and stable aggregates can be eluted. Thehistogram (Fig. 8) of the eluted NP distribution was asymmetric.

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Fig. 6 Chromatographic elution profiles of the DMEM-F12-FCS medium in the control run (dashed line) and in the presence of NPs at 60 mg ml�1 (solid line).Absorbance variations were monitored at 280 nm (grey lines), 480 nm (red lines) and 647 nm (blue lines) versus the eluted fractions.

Fig. 7 Fluorescence variations (expressed in %) for the A8–A14 eluted fractionsand calculated from (I0 � I)/I0 (ESI, Table S1†). I0 and I are the fluorescence of thedifferent fractions in the absence/presence of NPs. Alexa� 488-labeled BSA (>)and Alexa� 647-labeled IgG (C).

Fig. 8 NP distribution within the chromatographic fractions. Ce was quantifiedby ICP-MS.

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The A1 fraction corresponded to the dead volume of excludedproteins in the calibration run, as estimated by UV absorbance.The real “dead volume” was probably smaller than the corre-sponding A1 volume, but we did not collect the previous frac-tions. However, NP distribution was not strictly correlated withthe UV absorbance measurements: higher NP concentrationswere observed around the dead volume and in fractions with

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low absorbance (A1–A7) i.e. with a very small amount of protein.Fractions A1–A7 correspond mainly to very high molecularweight species, such as protein aggregates, and the amount ofNPs in these fractions is signicantly higher than in the otherfractions. It can be concluded that since the chromatogram at280 nm is not affected by the presence of CeO2 NPs, only CeO2

homo aggregates can be eluted in these fractions. This is inagreement with DLS experiments, showing aggregates largerthan hundreds of nanometers (Fig. 3b). From A8 to B5, thehistogram globally followed the absorbance registration. Weobserved that, for fractions A13–A14, where albumin was themain protein, the quantity of NPs was smaller than in fractionsA8–A10, where IgG was predominantly eluted. From B6 to C5,NPs were not detected.

Discussion

Mixing NPs in biological uid leads to strong NP surfacemodications. In most cases proteins adsorb onto the NPsurface by creating a corona.18 Protein adsorption onto surfacesis driven by various interactions such as van der Waal forces,Lewis acid forces, hydrogen bonds, electrostatic forces andmore entropic forces such as hydrophobic effects associatedwith water desorption from the solid supports.21 To date, mostof the published data concerning the NP protein corona wereobtained using NPs larger than 40 nm. Among all these studies,there is a great deal of evidence that NP–protein interactions areirreversible, with the creation of a hard protein corona (e.g. ref.22). However, modication of the sorption capacities as afunction of NP size has received less attention23 and theconclusions remain unclear. For large NPs (from 40–200 nm)the variation in size of NPs does not greatly modify adsorptionproperties. Cedervall et al. (2007)4 indicated that 200 nm parti-cles adsorbed a denser protein corona than 70 nm particles. Thenature of the adsorbed proteins may also differ as a function ofinitial particle size but these effects are not drastic. Nienhausalso evidenced a protein corona surrounding NPs smaller than


Fig. 9 Proposed and hypothetical aggregate structures.

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20 nm.24,25 But Casals et al. (2010)22 did not observe a hardprotein corona (surrounding 4 nm Au NPs).

The originality of our work was to investigate and describethe interactions of NPs and proteins of similar sizes (�7 to9 nm). In such a case, the NP surface cannot be considered to beinnite and at. Hence, the combination of Coulomb forcesbetween the proteins and the NPs as well as protein–proteinrepulsive forces can be at the origin of “so” (weak?) proteincorona formation instead of a “hard” corona.

Homoaggregation versus heteroaggregation

Homoaggregation of CeO2 NPs and more generally metal oxideNPs increases as a function of initial NP concentration, ionicstrength and pH in the iso-electric point (IEP) range. In thisstudy, the NP concentrations were chosen to t the concentra-tion ranges of classic cell toxicity assays. For such NP concen-trations, the circumneutral pH and high ionic strength of thenutritive media promote the homoaggregation of CeO2 NPswith aggregate sizes of several microns in DMEM-F12.

However when proteins (FCS) were added to the DMEM-F12,interactions between NPs and proteins occurred, leading todisaggregation of CeO2 NPs. The addition of proteins decreasedthe aggregate size to �38 nm (Fig. 5). This was a direct proof ofNP–protein interactions which were conrmed by zeta potentialmeasurements. At pH 7.4, the NPs are positively charged andthe proteins negatively charged (Fig. SI-4†). However the heter-oaggregates formed in the DMEM-F12-FCS are negativelycharged (Fig. SI-4†). This highlights the fact that that theproteins impose a negative zeta potential on heteroaggregatesand form an outer layer surrounding the NPs.

The uorescence analysis and CeO2 NP distribution withinthe separation domain of the chromatography column showedthat more stable interactions occur with IgG than BSA. A higherNP content was associated with the protein population in whichIgGs were mainly present. Conversely, and while BSA molarconcentration is ten times higher than IgG in serum, NPs werepresent in smaller amounts in the fraction in which BSA waspredominant. In parallel, a detailed analysis showed that onlyIgG uorescence signals were signicantly decreased in thefractions in which both labeled proteins were co-eluted. Basedon this analysis, we concluded that in both DMEM-F12-FCS andTris buffer, CeO2 NPs interactedmainly with IgG, even if the IgGconcentration was lower than that of BSA.

Reversibility of the NP–protein complex and hetero-aggregatestructure

Several examples of evidence of proteins forming a hard (irre-versible) corona around NPs are given in the literature (e.g.(ref. 9–11,14,18–22)), but our results may be puzzling as afunction of the size ratio between proteins and NPs. Our studyfound evidence that for a size ratio [protein–NPs] close to 1,interactions between NPs and proteins are reversible.

The rst evidence of this reversibility is given by size exclu-sion chromatography (Results, part 2). The Superose 6 gel phasechosen is known for its high porosity, and the separation range(5 � 103 to 5 � 106 Da) allows the elution of large aggregates.


Moreover, the agarose gel (main component of Superose 6) isslightly negatively charged at pH ¼ 7.4. Although this separa-tion method is mild and not supposed to disturb the NP–protein equilibrium,1 94% of cerium is retained inside the gelwhile all the proteins are eluted. This behavior indicates thatthe heteroaggregates formed in the DMEM-F12-FCS prior toinjection are strongly destabilised in the gel.

Aer 24 h in DMEM-F12-FCS, DLS studies demonstrated thataggregate sizes are split into two populations: a major fractioncentered on 38 nm and a minor fraction at 550 nm (Fig. 5).Based on our data, it is possible to hypothesize the structure ofthe 38 nm heteroaggregates (Fig. 9).

For instance, a compact aggregate (with hexagonal closepacking for instance) composed of one CeO2 NP surrounded byone protein layer (average size of one protein: 7 nm, see part 2for instance) would be composed of 12 proteins and have ahydrodynamic diameter of 21 nm (Fig. 9, model A). A secondlayer of proteins would lead to aggregates composed of 54proteins with a hydrodynamic radius of 35 nm (Fig. 9, model A).On the other hand, an aggregate with an open structure (Fig. 9,model B) could form 35–38 nm aggregates but with far fewerproteins (as for a linear aggregate composed of one CeO2 NPand 4 proteins). These two proposed structures correspond toextreme compactness (with high and low fractal dimensions).Such simple models only take into account attractive forcesbetween NPs and proteins but not the protein–protein repulsiveforces (exclusion volume). Therefore such highly compactsystems are not likely to be formed in nutritive media.

A more accurate structure of aggregates would be a unit baseformed by one CeO2 NP core with one layer of protein (Fig. 9,model C and D). This would lead to aggregates close to 38 nm.In such structures, each unit base is surrounded by fewer than12 proteins due to protein–protein repulsive forces leading tomolecular weights of 1000–3000 kDa for models C and Drespectively. With the hypothesis that NP–protein interactionsare irreversible, our proposed models C and D would have beeneluted in the A1–A6 fractions. Indeed their molecular weight

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Fig. 10 Evolution of the protein adsorption process with time on a large surface(A–C) (adapted from (ref. 26)) and on small NPs (D).

Fig. 11 Effect of particle surface curvature on the distance between attractivesites with proteins.

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remains in the separation range of the Superose 6 gel below5 � 106 kDa.

Therefore another scenario is needed to explain the sizeexclusion chromatography results. The absence of NP–proteinaggregates in the eluted fraction suggests that NP–Superose 6interactions are higher than NP–protein interactions. Thisxation of NPs onto the gel is certainly due to the slight negativecharge of the agarose at pH 7.4 (due to the pKa of the agarosefunctional group). Therefore competition for NP linkagesbetween the negative surface of the gel and the negativelycharged proteins is in favor of the gel. This result clearly showsthat the NP–protein interactions under our conditions arereversible.

The protein adsorption on an innite surface is related toCoulomb interactions between the proteins and the sorbent.Such interactions govern short term kinetics. When proteinsand NPs are approaching each other, Coulomb forces lead toeffective contact (Fig. 10A).26 At a short reaction time, only alimited number of attractive sites are involved. However,Coulomb forces decrease with the distance between charges(1/d2). Therefore, if attractive sites are close enough, new bondscan occur that will consequently modify the protein structure.The creation of new attractive bonds will lead to protein struc-ture rearrangement in a second step (Fig. 10B). Then, as afunction of the surface area of contact, entropically drivenmechanisms such as hydrophobic interactions and waterdesorption can occur (Fig. 10C). This latter interaction, occur-ring at a later time, is responsible for quasi irreversible proteinadsorption.

For proteins in contact with NPs of similar size, the reactionpathway is different due to the strong curvature effects. Indeed,the distance between attractive sites is longer than for a atsurface (Fig. 11). Considering spherical particles (7 and 40 nm)and proteins (7 nm) separated by a distance A–A0, the electro-static interactions between regularly distributed charges can becalculated at different positions (B–B0).27 R is the protein radius(3.5 nm), r is the distance between attractive sites on NPs and/or

1666 | Nanoscale, 2013, 5, 1658–1668

proteins. A–A0 was set at 0.4 nm to be coherent with theadsorption theories of polyelectrolytes.27 However, the surfacedistribution of attractive sites is more complex to estimate.Based on the low zeta potential of NP at pH 7.4 (+15 mV), weassume less than 2 or 1 mC cm�2 i.e. 0.12–0.06 charge per nm2

i.e. 1 charge for 8–16 nm2. Therefore r was set at 1.4 nm. Allconcluded that attractive forces between A and A0 (the nearestones) are �3 times higher than the force between B and B0 for40 nmNPs and�6 times higher for 7 nmNPs. Consequently, forthe 7 nm NPs, the attractive force between B and B0 will be toosmall to create a new bond between the protein and the NP.

In the case of very small NPs, so protein corona formationwould lead to different cell internalisation mechanismscompared with larger NPs. Indeed our experiment revealed lowstability of protein–NP heteroaggregates when injected inagarose gel. Agarose gel possesses typical characteristics thatresemble the composition of living tissues (rheological, surfaceproperties.) and has been widely used to simulate the trans-portation and release of molecules.28 Therefore our resultssuggest that the reversibility of the protein–NP interactionwould modify the mode of interaction between NPs and the cell,with possible desorption of the protein corona prior to contactwith membrane cells. However, in the case of larger particles itis more likely that the cell does not ‘see’ bare NPs. Since surfaceproperties are responsible for modication in the internal-isation process, it is possible that small NPs (similar in size toproteins) and larger NPs may be internalised by different cellmechanisms. It is obvious that further research is needed todene the limit of NP size between hard and so protein coronaformation.

Materials and methodsCeO2 particles

Nano-CeO2 particles were synthesized by aqueous precipitationof the Ce4+(NO3�)4 salt at acidic pH.29 Transmission electronmicroscopy and X-ray diffraction measurements have shownthat these nano-CeO2 are ellipsoidal cerianite crystallites with amean diameter of 7 nm. Their mean hydrodynamic diameter insolution is around 10 nm.30 The specic surface area (SSA) ofthese particles was evaluated at 400 m2 g�1.31 Their opticalproperties were measured by UV-vis spectrophotometry anduorescence spectroscopy.


Paper Nanoscale

Cell culture medium

Dulbecco's modied Eagle's medium-F12 (Gibco�) supple-mented with 10% fetal calf serum (DMEM-F12-FCS) was used inthis study.

Preparation of labeled proteins

Bovine serum albumin (BSA, SIGMA) and a mouse monoclonalantibody (IgG1 isotype) from our laboratory were labeled withuorescent dyes (Alexa Fluor� 488 and 647, Molecular Probes,Eugene, Oregon US) according to the manufacturers' instruc-tions. The dye/protein ratios were calculated to reach a signalintensity within the same range, taking into account theirrelative concentrations in serum (BSA around 40 mg ml�1 andIgG around 10 mg ml�1) and their molecular weights. The nalratios were 3 : 1 for BSA and 6 : 1 for IgG.

Preparation of sterile CeO2 NP suspensions

Aqueous suspensions of CeO2 NPs were prepared by dispersingnano-CeO2 powders in pure water (MilliQ, 18.2 U) at 10 mgml�1. The suspensions were passed through a 0.22 mm lter andcollected under a laminar ow hood.

Characterization of CeO2 NPs in solution

The behavior of NPs, with and without study proteins, inphysiological solution and cell culture media was analyzedusing dynamic light scattering (DLS). The measurements weremade using NanoZS (Malvern Instruments Inc, UK). Absorptionspectrophotometry (Carry 50) was used to determine the nalNP concentration. Aliquots (100 ml) were prepared to constitutea stock solution for all the tests, and bacteriological stability wascontrolled (chocolate agar method).

Circular dichroism spectroscopy (CD)

The CD spectra were recorded using a spectropolarimeter (J-810Jasco, France) equipped with a thermostatically controlled cellholder. An average of three scans were recorded at 25 �C using a1 mm cell, within the 190 to 290 nm wavelength range and abandwidth of 1 nm. The BSA and IgG concentrations were keptconstant at 3 mM and 2 mM, respectively, and NP concentrationswere varied from 10 to 60 mg ml�1.

Fluorescence spectroscopy measurement

Fluorescence signals were recorded according to NP addition.For unlabeled proteins, the excitation wavelength was set at280 nm and the emissions were collected within the 310–700 nm range. The uorescence of labeled proteins wascontrolled according to dye properties: the excitation wave-lengths of Alexa 488-BSA- and Alexa647-IgG- were monitored at488 and 647 nm and the emissions were monitored at 521 and674 nm, respectively. A constant lag time (1 minute) wasobserved before registration. The uorescence intensities weredetermined at the lemmax, and the data were plotted using theStern–Volmer representation:

I0/I ¼ f[NPs]


where I0 and I are the steady-state uorescence intensities in theabsence and presence of NPs, respectively.

Size exclusion chromatography

DMEM-F12-FCS was spiked with uorescently labeled BSA andIgG according to their physiological concentrations in serum,and avoiding any signicant dilution.

NP distribution within the cell culture medium and proteinprole analysis were performed by gel ltration chromatog-raphy using a Superose� 6 (GE Healthcare) packed column(separation range from 5 to 5000 kD, 2.6 � 30 cm) and high-performance liquid chromatography (AKTA Purier, GEHealthcare). The elution buffer was 8 mM Tris–HCl at pH 7.4and the chromatography processes were monitored by UVabsorbance at 280, 488 and 647 nm. Separation was performedat a constant ow rate of 22 cm h�1. Eluted fractions (4 ml) werecollected in sequence (from A1–A14, B12–B1 to C1–C5). Each ofthese was analyzed by UV/vis absorption and uorescencespectroscopy (Cary Eclipse, VARIAN, Agilent technologies).The NP content was determined by ICP-MS analysis (Agilent7700 series).

X-ray absorption spectroscopy

X-ray absorption spectroscopy (XAS) spectra were recorded intransmission mode on beamline 11.1 at the ELETTRAsynchrotron (Trieste, Italy). Spectra were acquired using anSi(111) monochromator above the Ce LIII-edge (5723 eV). Theion chambers for incident and transmitted beams were lledwith argon and nitrogen gas. XANES (X-ray absorption near edgestructure) data were obtained aer performing standardprocedures for pre-edge subtraction and normalization usingseveral programs developed by Michalowicz.32 EXAFS (extendedX-ray ne structure) data were obtained aer performing stan-dard procedures for pre-edge subtraction, normalization, poly-nomial removal and wave vector conversion using the IFEFFITsoware package.33 For each atomic shell, the interatomicdistance (R), coordination number (CN), and mean squareddisplacement (s2) were adjusted. The amplitude reductionfactor and the threshold position were tted to data fromreference compounds (CeO2) and xed for all subsequentanalyses.

Aer BSA–CeO2 and IgG–CeO2 interactions, samples werefreeze dried. The quantity of Ce was calculated so that the nalconcentration in the dried samples was greater than 1000 ppm,for detection on the Elettra XAS beamline. The samples weremixed with BN and pressed into pellets of homogeneousthickness.

Acknowledgements

The authors gratefully acknowledge Luca Olivi and his teamfrom the Elettra synchrotron (Italy) for their technical helpduring X-ray absorption spectroscopy experiments, and AgnesHagege for fruitful discussions. Financial support was providedby the Antiopes Network through the Grenelle Funding (French

Nanoscale, 2013, 5, 1658–1668 | 1667

Nanoscale Paper

Ministry “de l'ecologie, du Developpement durable” INERIS)and the ‘Impecnano’ Project.

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protein corona formation for nanomaterials and proteins of a similar size: hard or soft corona?

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